Microbial Mufanfs 4S 



(DHS/DHQ=15) heavily in favor of DHS. The activity 

 of a wild-type extract is approximately that required to 

 account for the rate of aromatic biosynthesis implied by 

 the observed growth rate of the culture. 



Two aspects of this study are particularly relevant to 

 our interests here. The first is the evidence that the reac- 

 tion involves a single enzymatic step. Since the reaction 

 is a dehydration, resembling the reactions catalyzed by 

 fumarase and aconitase, it would be expected, like the 

 latter two reactions, to involve a single enzyme; the only 

 plausible alternative would seem to be a series of reactions 

 involving interaction with a cofactor (for example, con- 

 jugation or change in level of oxidation), followed by 

 dehydration and then reversal of the interaction with the 

 cofactor. But no cofactor requirement, organic or inor- 

 ganic, could be demonstrated. The enzyme has been puri- 

 fied about tenfold by treatment with MnCl2, (NH4)2S04, 

 and calcium phosphate gel. The product thus obtained, 

 even after treatment with an anion-exchange resin to re- 

 move bound cofactors, was active without cofactor addi- 

 tion, and was equally active in tris-(hydroxymethyl)-amino- 

 methane hydrochloride buffer or in potassium phosphate 

 buffer. Hence, even though 5-dehydroquinase is still far 

 from crystalline, its lack of a demonstrable cofactor re- 

 quirement furnishes strong evidence that the reaction is 

 mediated by a single enzyme. 



The other aspect of this work that I should like to men- 

 tion is that even though dehydroquinase activity is regu- 

 larly demonstrable in extracts of the wild type, no activity 

 could be demonstrated in extracts of mutants that were 

 blocked in this reaction. The sensitivity of the assay was 

 such that one would have detected the enzyme in a con- 

 centration 1/300 that of the wild type. Furthermore, mix- 

 tures of wild-type and mutant extracts showed precisely 

 the same activity as the wild-type extract alone. This find- 

 ing allows one to reject the possibility that the inactivity 



